Norovirus vaccine formulations

ABSTRACT

The present invention relates to antigenic and vaccine compositions comprising Norovirus antigens and adjuvants, in particular, mixtures of monovalent VLPs and mixtures of multivalent VLPs, and to a process for the production of both monovalent and multivalent VLPs, the VLPs comprising capsid proteins from one or more Norovirus genogroups.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/330,854, filed Dec. 20, 2011, which is a continuation of U.S. patentapplication Ser. No. 12/816,495, filed Jun. 16, 2010, which is acontinuation of U.S. patent application Ser. No. 12/093,921, filed May15, 2008, which issued as U.S. Pat. No. 7,955,603 on Jun. 7, 2011, whichis a national stage application of International Application No.PCT/US2007/079929, filed Sep. 28, 2007, which claims priority to U.S.Patent Application Ser. No. 60/847,912, filed Sep. 29, 2006, and U.S.Patent Application Ser. No. 60/973,392, filed Sep. 18, 2007, all ofwhich are herein incorporated by reference in their entireties.

STATEMENT OF GOVERNMENT SUPPORT

This invention was produced with government support from the US ArmyMedical Research and Material Command, under contract numbersDAMD17-01-C-0400 and W81XWH-05-C-0135. The government may have certainrights to the invention.

FIELD OF THE INVENTION

The invention is in the field of vaccines, particularly vaccines forNoroviruses. In addition, the invention relates to methods of preparingvaccine compositions and methods of inducing an immunogenic response.

BACKGROUND OF THE INVENTION

Noroviruses are non-cultivatable human Caliciviruses that have emergedas the single most important cause of epidemic outbreaks of nonbacterialgastroenteritis (Glass et al., 2000; Hardy et al., 1999). The clinicalsignificance of Noroviruses was under-appreciated prior to thedevelopment of sensitive molecular diagnostic assays. The cloning of theprototype genogroup I Norwalk virus (NV) genome and the production ofvirus-like particles (VLPs) from a recombinant Baculovirus expressionsystem led to the development of assays that revealed widespreadNorovirus infections (Jiang et al. 1990; 1992).

Noroviruses are single-stranded, positive sense RNA viruses that containa non-segmented RNA genome. The viral genome encodes three open readingframes, of which the latter two specify the production of the majorcapsid protein and a minor structural protein, respectively (Glass etal. 2000). When expressed at high levels in eukaryotic expressionsystems, the capsid protein of NV, and certain other Noroviruses,self-assembles into VLPs that structurally mimic native Norovirusvirions. When viewed by transmission electron microscopy, the VLPs aremorphologically indistinguishable from infectious virions isolated fromhuman stool samples.

Immune responses to Noroviruses are complex, and the correlates ofprotection are just now being elucidated. Human volunteer studiesperformed with native virus demonstrated that mucosally-derived memoryimmune responses provided short-term protection from infection andsuggested that vaccine-mediated protection is feasible (Lindesmith etal. 2003; Parrino et al. 1997; Wyatt et al., 1974).

Although Norovirus cannot be cultivated in vitro, due to theavailability of VLPs and their ability to be produced in largequantities, considerable progress has been made in defining theantigenic and structural topography of the Norovirus capsid. VLPspreserve the authentic confirmation of the viral capsid protein whilelacking the infectious genetic material. Consequently, VLPs mimic thefunctional interactions of the virus with cellular receptors, therebyeliciting an appropriate host immune response while lacking the abilityto reproduce or cause infection. In conjunction with the NIH, BaylorCollege of Medicine studied the humoral, mucosal and cellular immuneresponses to NV VLPs in human volunteers in an academic,investigator-sponsored Phase I clinical trial. Orally administered VLPswere safe and immunogenic in healthy adults (Ball et al. 1999; Tacket etal. 2003). At other academic centers, preclinical experiments in animalmodels have demonstrated enhancement of immune responses to VLPs whenadministered intranasally with bacterial exotoxin adjuvants (Guerrero etal. 2001; Nicollier-Jamot et al. 2004; Periwal et al. 2003).Collectively, these data suggest that a vaccine consisting of properlyformulated VLPs represents a viable strategy to immunize againstNorovirus infection.

SUMMARY OF THE INVENTION

The present invention provides antigenic and vaccine formulationscomprising a Norovirus antigen. In one embodiment, the formulationsfurther comprise at least one adjuvant. The Norovirus antigen can bederived from genogroup I or genogroup II viral sequences or a consensusviral sequence. The Norovirus formulations comprise antigenic peptides,proteins or virus-like particles (VLPs). In one embodiment, the VLPs maybe denatured. In another embodiment, the antigenic peptides and proteinsare selected from the group consisting of capsid monomers, capsidmultimers, protein aggregates, and mixtures thereof. In anotherembodiment, the Norovirus antigen is present in a concentration fromabout 0.01% to about 80% by weight. The dosage of Norovirus antigen ispresent in an amount from about 1 μg to about 100 mg per dose.

In another embodiment, the Norovirus VLPs are recombinant VLPs producedin an expression system using a Norovirus nucleic acid sequence, whichencodes at least one capsid protein or fragment thereof. The capsidprotein is selected from the group consisting of VP1 and VP2 or acombination thereof. The expression system can be a recombinant cellularexpression system such as a yeast, bacterial, insect, mammalianexpression system, or a baculovirus-infected cellular expression system.

In still another embodiment, the composition further comprises adelivery agent, which functions to enhance antigen uptake by providing adepot effect, increase antigen retention time at the site of delivery,or enhance the immune response through relaxation of cellular tightjunctions at the delivery site. The delivery agent can be a bioadhesive,preferably a mucoadhesive selected from the group consisting ofglycosaminoglycans (e.g., chondroitin sulfate, dermatan sulfatechondroitin, keratan sulfate, heparin, heparan sulfate, hyaluronan),carbohydrate polymers (e.g., pectin, alginate, glycogen, amylase,amylopectin, cellulose, chitin, stachyose, unulin, dextrin, dextran),cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol,polyvinyl pyrollidone, polysaccharides (including mucin and othermucopolysaccharides)cellulose derivatives (e.g., hydroxypropylmethylcellulose, carboxymethylcellulose), proteins (e.g. lectins,fimbrial proteins), and deoxyribonucleic acid. Preferably, themucoadhesive is a polysaccharide. More preferably, the mucoadhesive ischitosan, or a mixture containing chitosan, such as a chitosan salt orchitosan base.

In yet another embodiment, the present invention provides a compositionfurther comprising an adjuvant. The adjuvant may be selected from thegroup consisting of toll-like receptor (TLR) agonists, monophosphoryllipid A (MPL®), synthetic lipid A, lipid A mimetics or analogs, aluminumsalts, cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpGoligos, lipopolysaccharide (LPS) of gram-negative bacteria,polyphosphazenes, emulsions, virosomes, cochleates,poly(lactide-co-glycolides) (PLG) microparticles, poloxamer particles,microparticles, endotoxins, for instance bacterial endotoxins andliposomes. Preferably, the adjuvant is a toll-like receptor (TLR)agonist. More preferably, the adjuvant is MPL®.

The compositions of the present invention may be provided as a liquidformulation or a dry powder formulation. Dry power formulations of thepresent invention may contain an average particle size from about 10 toabout 500 micrometers in diameter. In one embodiment, the composition isan antigenic formulation. In another embodiment, the composition isformulated for administration as a vaccine. Suitable routes ofadministration include mucosal, intramuscular, intravenous,subcutaneous, intradermal, subdermal, or transdermal. In particular, theroute of administration may be intramuscular or mucosal, with preferredroutes of mucosal administration including intranasal, oral, or vaginalroutes of administration. In another embodiment, the composition isformulated as a nasal spray, nasal drops, or dry powder, wherein theformulation is administered by rapid deposition within the nasal passagefrom a device containing the formulation held close to or inserted intothe nasal passageway. In another embodiment, the formulation isadministrated to one or both nostrils.

The present invention also provides methods for generating an immuneresponse to Norovirus in a subject, comprising administering to thesubject an antigenic formulation or a vaccine comprising the Noroviruscomposition. In one embodiment, the antigenic formulations and vaccinescomprising the Norovirus composition find use in generating antibodiesto one or more Norovirus antigens. In another embodiment, the Norovirusvaccine formulations may be used to treat Norovirus infections.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an in vitro antigen-specific proliferation assay ofmurine cervical lymph node cells following in vivo intranasalimmunization with 10 μg VLP.

FIG. 2 illustrates in vitro antigen-specific proliferation assay ofsplenocytes following in vivo intranasal immunization with 10 μg VLP.

FIG. 3 illustrates in vitro antigen-specific proliferation assay ofsplenocytes following in vivo intraperitoneal immunization with 25 μgVLP.

FIG. 4 illustrates VLP-specific IgG or IgA from antibody secreting cells(ASCs) measured by ELISPOT assay.

FIG. 5 illustrates VLP-specific IgG measured by ELISA.

FIG. 6 illustrates the result of a potency assay for serum IgG responseagainst Norwalk VLPs.

FIG. 7 depicts the results of a potency assay comparing serum IgGresponses against Norwalk VLPs in mice immunized with either a liquidformulation of the antigen or a formulation reconstituted from drypowder. The graph shows potency versus concentration of Norwalk VLPs inthe different formulations.

FIG. 8 shows the serum IgG response in rabbits on day 21 (left panel)and day 42 (right panel) following administration of differentformulations of Norovirus VLP vaccine.

FIG. 9 illustrates the serum IgG response in rabbits immunizedintranasally with either a liquid formulation or a dry powderformulation of Norwalk VLPs.

FIG. 10 depicts the stability of dry powder formulation as measured byquantitative SDS-PAGE analysis and size exclusion chromatography (SEC).Regression analysis indicates no statistical trends in either the totalor intact μg VLP per 10 mg dry powder over 1 year. The percent aggregateis a calculation assuming that VLP protein not detected by SEC, comparedto the total VLP protein by quantitative SDS-PAGE, is aggregated.

FIG. 11 illustrates the results of an ELISA assay of anti-Norovirusantibody response in mice immunized i.p. with multiple Norovirusantigens. The thin arrows indicate booster injections with formulationscontaining only Norwalk VLPs. The thick arrows denote booster injectionswith formulations containing both Norwalk and Houston VLPs.

FIG. 12 illustrates an ELISA assay of anti-Norovirus antibody responsein mice immunized i.p. with either Norwalk VLPs, Houston VLPs, or acombination of Norwalk and Houston VLPs.

FIG. 13 shows the presence of Norwalk VLP-specific long-lived plasmacells in splenocytes (A), cervical lymph nodes (B), and bone marrow (C)in mice 114 days after intranasal immunization with Norwalk VLPs inmice.

FIG. 14 depicts the Norwalk-specific memory B cell response insplenocytes of mice immunized intranasally with Norwalk VLPs. Panel Ashows IgA antibody secreting cells on day 0 (left graph) and day 4 inculture with Norwalk VLPs (right graph). Panel B shows the IgG antibodysecreting cells on day 0 (left graph) and day 4 in culture with NorwalkVLPs (right graph). The difference in the number of cells between day 0and day 4 indicates the level of memory B cell expansion anddifferentiation.

FIG. 15 shows the ELISPOT assay results of peripheral blood mononuclearcells isolated from rabbits immunized intranasally with a Norwalk VLPvaccine formulation. The left panel shows the number of NorwalkVLP-specific antigen secreting cells (ASCs) at day 0 (day of tissueharvest), while the right panel illustrates the number of NorwalkVLP-specific ASCs after 4 days in culture with Norwalk VLPs. Thedifference in the number of cells between day 0 and day 4 indicates thememory B cell response.

FIG. 16 shows the ELISPOT assay results of splenocytes harvested fromrabbits immunized intranasally with a Norwalk VLP vaccine formulation.The left panel shows the number of Norwalk VLP-specific antigensecreting cells (ASCs) at day 0 (day of tissue harvest), while the rightpanel illustrates the number of Norwalk VLP-specific ASCs after 4 daysin culture with Norwalk VLPs. The difference in the number of cellsbetween day 0 and day 4 indicates the memory B cell response.

FIG. 17 shows the ELISPOT assay results of bone marrow cells harvestedfrom the tibias of rabbits immunized intranasally with a Norwalk VLPvaccine formulation. The left panel shows the number of NorwalkVLP-specific antigen secreting cells (ASCs) at day 0 (day of tissueharvest), while the right panel illustrates the number of NorwalkVLP-specific ASCs after 4 days in culture with Norwalk VLPs. Thepresence of ASCs at day 0 indicates the presence of long-lived plasmacells. The difference in the number of cells between day 0 and day 4indicates the memory B cell response.

FIG. 18 shows the ELISPOT assay results of mesenteric lymph node cellsharvested from rabbits immunized intranasally with a Norwalk VLP vaccineformulation. Panel A shows IgG positive antibody secreting cells (ASCs)specific for Norwalk VLPs. Panel B shows IgA positive ASCs specific forNorwalk VLPs. The left panels show the number of Norwalk VLP-specificASCs at day 0 (day of tissue harvest), while the right panels illustratethe number of Norwalk VLP-specific ASCs after 4 days in culture withNorwalk VLPs. The presence of ASCs at day 0 indicates the presence oflong-lived plasma cells. The difference in the number of cells betweenday 0 and day 4 indicates the memory B cell response.

FIG. 19 illustrates in vitro antigen-specific proliferation assay ofsplenocytes following in vivo intranasal immunization in rabbits. Theleft panel shows T cell proliferation upon restimulation with NorwalkVLPs in unfractionated splenocytes, while the right panel shows CD4+ Tcell proliferation upon restimulation with Norwalk VLPs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to Norovirus antigenic and vaccinecompositions and methods of preparing the compositions. In particular,the present invention provides a composition that comprises a Norovirusantigen and at least one adjuvant. Additionally or alternatively, thecomposition may further comprise at least one delivery agent. Theinvention also provides methods of administering the composition to ananimal to produce an immune response or generate antibodies to Norovirusantigens.

Norovirus Antigens

The invention provides a composition comprising one or more Norovirusantigens. By “Norovirus,” “Norovirus (NOR),” “norovirus,” andgrammatical equivalents herein, are meant members of the genus Norovirusof the family Caliciviridae. In some embodiments, a Norovirus caninclude a group of related, positive-sense single-stranded RNA,nonenveloped viruses that can be infectious to human or non-humanmammalian species. In some embodiments, a Norovirus can cause acutegastroenteritis in humans. Noroviruses also can be referred to as smallround structured viruses (SRSVs) having a defined surface structure orragged edge when viewed by electron microscopy. Included within theNoroviruses are at least four genogroups (GI-IV) defined by nucleic acidand amino acid sequences, which comprise 15 genetic clusters. The majorgenogroups are GI and GII. GIII and GIV are proposed but generallyaccepted. Representative of GIII is the bovine, Jena strain. GIVcontains one virus, Alphatron, at this time. For a further descriptionof Noroviruses see Vinje et al. J. Clin. Micro. 41:1423-1433 (2003). By“Norovirus” also herein is meant recombinant Norovirus virus-likeparticles (rNOR VLPs). In some embodiments, the recombinant NorovirusVLPs are produced in an expression system using a Norovirus nucleic acidsequence, which encodes at least one capsid protein or fragment thereof.In other embodiments, recombinant expression of at least the Noroviruscapsid protein encoded by ORF2 in cells, e.g., from a baculovirus vectorin Sf9 cells, can result in spontaneous self-assembly of the capsidprotein into VLPs. In yet other embodiments, recombinant expression ofat least the Norovirus proteins encoded by ORF1 and ORF2 in cells, e.g.,from a baculovirus vector in Sf9 cells, can result in spontaneousself-assembly of the capsid protein into VLPs. The Norovirus nucleicacid sequence may also be a consensus sequence comprising variousNorovirus strains or a synthetic construct modified to enhance yields orstability, or improve antigenic or immunogenic properties of the encodedantigen. VLPs are structurally similar to Noroviruses but lack the viralRNA genome and therefore are not infectious. Accordingly, “Norovirus”includes virions that can be infectious or non-infectious particles,which include defective particles.

Non-limiting examples of Noroviruses include Norwalk virus (NV, GenBankM87661, NP₀₅₆₈₂₁), Southampton virus (SHV, GenBank L07418), DesertShield virus (DSV, U04469), Hesse virus (HSV), Chiba virus (CHV, GenBankAB042808), Hawaii virus (HV, GenBank U07611), Snow Mountain virus (SMV,GenBank U70059), Toronto virus (TV, Leite et al., Arch. Virol.141:865-875), Bristol virus (BV), Jena virus (JV, AJ01099), Marylandvirus (MV, AY032605), Seto virus (SV, GenBank AB031013), Camberwell (CV,AF145896), Lordsdale virus (LV, GenBank X86557), Grimsby virus (GrV,AJ004864), Mexico virus (MXV, GenBank U22498), Boxer (AF538679), C59(AF435807), VA115 (AY038598), BUDS (AY660568), Houston virus (HoV),Minerva strain (EF126963.1), Laurens strain (EF126966.1), MOH(AF397156), Parris Island (PiV; AY652979), VA387 (AY038600), VA207(AY038599), and Operation Iraqi Freedom (OIF, AY675554). The nucleicacid and corresponding amino acid sequences of each are all incorporatedby reference in their entirety. In some embodiments, a cryptogram can beused for identification purposes and is organized: host species fromwhich the virus was isolated/genus abbreviation/speciesabbreviation/strain name/year of occurrence/country of origin. (Green etal., Human Caliciviruses, in Fields Virology Vol. 1 841-874 (Knipe andHowley, editors-in-chief, 4th ed., Lippincott Williams & Wilkins 2001)).Use of a combination of Norovirus genogroups such as a genogroup I.1(Norwalk virus) and II.4 (Houston virus) or other commonly circulatingstrains, or synthetic constructs representing combinations or portionsthereof are preferred in some embodiments. New strains of Norovirusesare routinely identified (Centers for Disease Control, Morbidity andMortality Weekly Report, 56(33):842-846 (2007)) and consensus sequencesof two or more viral strains may also be used to express Norovirusantigens.

The Norovirus antigen may be in the form of peptides, proteins, orvirus-like particles (VLPs). In a preferred embodiment, the Norovirusantigen comprises VLPs. As used herein, “virus-like particle(s) or VLPs”refer to a virus-like particle(s), fragment(s), aggregates, orportion(s) thereof produced from the capsid protein coding sequence ofNorovirus and comprising antigenic characteristic(s) similar to those ofinfectious Norovirus particles. Norovirus antigens may also be in theform of capsid monomers, capsid multimers, protein or peptide fragmentsof VLPs, or aggregates or mixtures thereof. The Norovirus antigenicproteins or peptides may also be in a denatured form, produced usingmethods known in the art.

Norovirus antigens may also include variants of the said capsid proteinsor fragments thereof expressed on or in the VLPs of the invention. Thevariants may contain alterations in the amino acid sequences of theconstituent proteins. The term “variant” with respect to a polypeptiderefers to an amino acid sequence that is altered by one or more aminoacids with respect to a reference sequence. The variant can have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. Alternatively, a variant can have “nonconservative” changes,e.g., replacement of a glycine with a tryptophan. Analogous minorvariations can also include amino acid deletion or insertion, or both.Guidance in determining which amino acid residues can be substituted,inserted, or deleted without eliminating biological or immunologicalactivity can be found using computer programs well known in the art, forexample, DNASTAR software.

General texts which describe molecular biological techniques, which areapplicable to the present invention, such as cloning, mutation, and thelike, include Berger and Kimmel, Guide to Molecular Cloning Techniques,Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif.(Berger); Sambrook et al., Molecular Cloning—A Laboratory Manual (3rdEd.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,2000 (“Sambrook”) and Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (“Ausubel”).These texts describe mutagenesis, the use of vectors, promoters and manyother relevant topics related to, e.g., the cloning and mutating ofcapsid proteins of Norovirus. Thus, the invention also encompasses usingknown methods of protein engineering and recombinant DNA technology toimprove or alter the characteristics of the proteins expressed on or inthe VLPs of the invention. Various types of mutagenesis can be used toproduce and/or isolate variant nucleic acids including concensussequences that encode for protein molecules and/or to furthermodify/mutate the proteins in or on the VLPs of the invention. Theyinclude but are not limited to site-directed, random point mutagenesis,homologous recombination (DNA shuffling), mutagenesis using uracilcontaining templates, oligonucleotide-directed mutagenesis,phosphorothioate-modified DNA mutagenesis, mutagenesis using gappedduplex DNA or the like. Additional suitable methods include pointmismatch repair, mutagenesis using repair-deficient host strains,restriction-selection and restriction-purification, deletionmutagenesis, mutagenesis by total gene synthesis, double-strand breakrepair, and the like.

The VLPs of the present invention can be formed from either the fulllength Norovirus capsid protein such as VP1 and/or VP2 proteins orcertain VP1 or VP2 derivatives using standard methods in the art.Alternatively, the capsid protein used to form the VLP is a truncatedcapsid protein. In some embodiments, for example, at least one of theVLPs comprises a truncated VP1 protein. In other embodiments, all theVLPs comprise truncated VP1 proteins. The truncation may be an N- orC-terminal truncation. Truncated capsid proteins are suitably functionalcapsid protein derivatives. Functional capsid protein derivatives arecapable of raising an immune response (if necessary, when suitablyadjuvanted) in the same way as the immune response is raised by a VLPconsisting of the full length capsid protein.

VLPs may contain major VP1 proteins and/or minor VP2 proteins.Preferably each VLP contains VP1 and/or VP2 protein from only oneNorovirus genogroup giving rise to a monovalent VLP. As used herein, theterm “monovalent” means the antigenic proteins are derived from a singleNorovirus genogroup. For example, the VLPs contain VP1 and/or VP2 from avirus strain of genogroup I (e.g., VP1 and VP2 from Norwalk virus).Preferably the VLP is comprised of predominantly VP1 proteins. In oneembodiment of the invention, the antigen is a mixture of monovalent VLPswherein the composition includes VLPs comprised of VP1 and/or VP2 from asingle Norovirus genogroup mixed with VLPs comprised of VP1 and/or VP2from a different Norovirus genogroup taken from multiple viral strains(e.g. Norwalk virus and Houston virus). Purely by way of example thecomposition can contain monovalent VLPs from one or more strains ofNorovirus genogroup I together with monovalent VLPs from one or morestrains of Norovirus genogroup II. Preferably, the Norovirus VLP mixtureis composed of the strains of Norwalk and Houston Noroviruses.

However, in an alternative embodiment of the invention, the VLPs may bemultivalent VLPs that comprise, for example, VP1 and/or VP2 proteinsfrom one Norovirus genogroup intermixed with VP1 and/or VP2 proteinsfrom a second Norovirus genogroup, wherein the different VP1 and VP2proteins are not chimeric VP1 and VP2 proteins, but associate togetherwithin the same capsid structure to form immunogenic VLPs. As usedherein, the term “multivalent” means that the antigenic proteins arederived from two or more Norovirus genogroups. Multivalent VLPs maycontain VLP antigens taken from two or more viral strains. Purely by wayof example the composition can contain multivalent VLPs comprised ofcapsid monomers or multimers from one or more strains of Norovirusgenogroup I together with capsid monomers or multimers from one or morestrains of Norovirus genogroup II. Preferably, the multivalent VLPscontain capsid proteins from the strains of Norwalk and HoustonNoroviruses.

The combination of monovalent or multivalent VLPs within the compositionpreferably would not block the immunogenicity of each VLP type. Inparticular it is preferred that there is no interference betweenNorovirus VLPs in the combination of the invention, such that thecombined VLP composition of the invention is able to elicit immunityagainst infection by each Norovirus genotype represented in the vaccine.Suitably the immune response against a given VLP type in the combinationis at least 50% of the immune response of that same VLP type whenmeasured individually, preferably 100% or substantially 100%. The immuneresponse may suitably be measured, for example, by antibody responses,as illustrated in the examples herein.

Multivalent VLPs may be produced by separate expression of theindividual capsid proteins followed by combination to form VLPs.Alternatively multiple capsid proteins may be expressed within the samecell, from one or more DNA constructs. For example, multiple DNAconstructs may be transformed or transfected into host cells, eachvector encoding a different capsid protein. Alternatively a singlevector having multiple capsid genes, controlled by a shared promoter ormultiple individual promoters, may be used. IRES elements may also beincorporated into the vector, where appropriate. Using such expressionstrategies, the co-expressed capsid proteins may be co-purified forsubsequent VLP formation, or may spontaneously form multivalent VLPswhich can then be purified.

A preferred process for multivalent VLP production comprises preparationof VLP capsid proteins or derivatives, such as VP1 and/or VP2 proteins,from different Norovirus genotypes, mixing the proteins, and assembly ofthe proteins to produce multivalent VLPs. The capsid proteins may be inthe form of a crude extract, be partially purified or purified prior tomixing. Assembled monovalent VLPs of different genogroups may bedisassembled, mixed together and reassembled into multivalent VLPs.Preferably the proteins or VLPs are at least partially purified beforebeing combined. Optionally, further purification of the multivalent VLPsmay be carried out after assembly.

Suitably the VLPs of the invention are made by disassembly andreassembly of VLPs, to provide homogenous and pure VLPs. In oneembodiment multivalent VLPs may be made by disassembly of two or moreVLPs, followed by combination of the disassembled VLP components at anysuitable point prior to reassembly. This approach is suitable when VLPsspontaneously form from expressed VP1 protein, as occurs for example, insome yeast strains. Where the expression of the VP1 protein does notlead to spontaneous VLP formation, preparations of VP1 proteins orcapsomers may be combined before assembly into VLPs.

Where multivalent VLPs are used, preferably the components of the VLPsare mixed in the proportions in which they are desired in the finalmixed VLP. For example, a mixture of the same amount of a partiallypurified VP1 protein from Norwalk and Houston viruses (or otherNorovirus strains) provides a multivalent VLP with approximately equalamounts of each protein.

Compositions comprising multivalent VLPs may be stabilized by solutionsknown in the art, such as those of WO 98/44944, WO0045841, incorporatedherein by reference.

Compositions of the invention may comprise other proteins or proteinfragments in addition to VP1 and VP2 proteins or derivatives. Otherproteins or peptides may also be co-administered with the composition ofthe invention. Optionally the composition may also be formulated orco-administered with non-Norovirus antigens. Suitably these antigens canprovide protection against other diseases.

The VP1 protein or functional protein derivative is suitably able toform a VLP, and VLP formation can be assessed by standard techniquessuch as, for example, electron microscopy and dynamic laser lightscattering.

Antigen Preparation

The antigenic molecules of the present invention can be prepared byisolation and purification from the organisms in which they occurnaturally, or they may be prepared by recombinant techniques. Preferablythe Norovirus VLP antigens are prepared from insect cells such as Sf9 orH5 cells, although any suitable cells such as E. coli or yeast cells,for example, S. cerevisiae, S. pombe, Pichia pastori or other Pichiaexpression systems, mammalian cell expression such as CHO or HEK systemsmay also be used. When prepared by a recombinant method or by synthesis,one or more insertions, deletions, inversions or substitutions of theamino acids constituting the peptide may be made. Each of theaforementioned antigens is preferably used in the substantially purestate.

The procedures of production of norovirus VLPs in insect cell culturehave been previously disclosed in U.S. Pat. No. 6,942,865, which isincorporated herein by reference in its entirety. Briefly, a cDNA fromthe 3′ end of the genome containing the viral capsid gene (ORF2) and aminor structural gene (ORF3) were cloned. The recombinant baculovirusescarrying the viral capsid genes were constructed from the cloned cDNAs.Norovirus VLPs were produced in Sf9 or H5 insect cell cultures.

Adjuvants

The invention further provides a composition comprising adjuvants foruse with the Norovirus antigen. Most adjuvants contain a substancedesigned to protect the antigen from rapid catabolism, such as aluminumhydroxide or mineral oil, and a stimulator of immune responses, such asBordatella pertussis or Mycobacterium tuberculosis derived proteins.Suitable adjuvants are commercially available as, for example, Freund'sIncomplete Adjuvant and Complete Adjuvant (Pifco Laboratories, Detroit,Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.);aluminum salts such as aluminum hydroxide gel (alum) or aluminumphosphate; salts of calcium, iron or zinc; an insoluble suspension ofacylated tyrosine; acylated sugars; cationically or anionicallyderivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; and Quil A.

Suitable adjuvants also include, but are not limited to, toll-likereceptor (TLR) agonists, monophosphoryl lipid A (MPL), synthetic lipidA, lipid A mimetics or analogs, aluminum salts, cytokines, saponins,muramyl dipeptide (MDP) derivatives, CpG oligos, lipopolysaccharide(LPS) of gram-negative bacteria, polyphosphazenes, emulsions, virosomes,cochleates, poly(lactide-co-glycolides) (PLG) microparticles, poloxamerparticles, microparticles, and liposomes. Preferably, the adjuvants arenot bacterially-derived exotoxins. Preferred adjuvants are those whichstimulate a Th1 type response such as 3DMPL or QS21.

Monophosphoryl Lipid A (MPL), a non-toxic derivative of lipid A fromSalmonella, is a potent TLR-4 agonist that has been developed as avaccine adjuvant (Evans et al. 2003). In pre-clinical murine studiesintranasal MPL has been shown to enhance secretory, as well as systemic,humoral responses (Baldridge et al. 2000; Yang et al. 2002). It has alsobeen proven to be safe and effective as a vaccine adjuvant in clinicalstudies of greater than 120,000 patients (Baldrick et al., 2002; 2004).MPL stimulates the induction of innate immunity through the TLR-4receptor and is thus capable of eliciting nonspecific immune responsesagainst a wide range of infectious pathogens, including both gramnegative and gram positive bacteria, viruses, and parasites (Baldrick etal. 2004; Persing et al. 2002). Inclusion of MPL in intranasalformulations should provide rapid induction of innate responses,eliciting nonspecific immune responses from viral challenge whileenhancing the specific responses generated by the antigenic componentsof the vaccine.

Accordingly, in one embodiment, the present invention provides acomposition comprising monophosphoryl lipid A (MPL®) or 3 De-O-acylatedmonophosphoryl lipid A (3D-MPL®) as an enhancer of adaptive and innateimmunity. Chemically 3D-MPL® is a mixture of 3 De-O-acylatedmonophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred formof 3 De-O-acylated monophosphoryl lipid A is disclosed in EuropeanPatent 0 689 454 B1 (SmithKline Beecham Biologicals SA), which isincorporated herein by reference. In another embodiment, the presentinvention provides a composition comprising synthetic lipid A, lipid Amimetics or analogs, such as BioMira's PET Lipid A, or syntheticderivatives designed to function like TLR-4 agonists.

The term “effective adjuvant amount” or “effective amount of adjuvant”will be well understood by those skilled in the art, and includes anamount of one or more adjuvants which is capable of stimulating theimmune response to an administered antigen, i.e., an amount thatincreases the immune response of an administered antigen composition, asmeasured in terms of the IgA levels in the nasal washings, serum IgG orIgM levels, or B and T-Cell proliferation. Suitably effective increasesin immunoglobulin levels include by more than 5%, preferably by morethan 25%, and in particular by more than 50%, as compared to the sameantigen composition without any adjuvant.

Delivery Agent

The invention also provides a composition comprising a delivery agentwhich functions to enhance antigen uptake based upon, but not restrictedto, increased fluid viscosity due to the single or combined effect ofpartial dehydration of host mucopolysaccharides, the physical propertiesof the delivery agent, or through ionic interactions between thedelivery agent and host tissues at the site of exposure, which providesa depot effect. Alternatively, the delivery agent can increase antigenretention time at the site of delivery (e.g., delay expulsion of theantigen). Such a delivery agent may be a bioadhesive agent. Inparticular, the bioadhesive may be a mucoadhesive agent selected fromthe group consisting of glycosaminoglycans (e.g., chondroitin sulfate,dermatan sulfate chondroitin, keratan sulfate, heparin, heparan sulfate,hyaluronan), carbohydrate polymers (e.g., pectin, alginate, glycogen,amylase, amylopectin, cellulose, chitin, stachyose, unulin, dextrin,dextran), cross-linked derivatives of poly(acrylic acid), polyvinylalcohol, polyvinyl pyrollidone, polysaccharides (including mucin andother mucopolysaccharides) cellulose derivatives (e.g., hydroxypropylmethylcellulose, carboxymethylcellulose), proteins (e.g. lectins,fimbrial proteins), and deoxyribonucleic acid. Preferably, themucoadhesive agent is a polysaccharide, such as chitosan, a chitosansalt, or chitosan base (e.g. chitosan glutamate).

Chitosan, a positively charged linear polysaccharide derived from chitinin the shells of crustaceans, is a bioadhesive for epithelial cells andtheir overlaying mucus layer. Formulation of antigens with chitosanincreases their contact time with the nasal membrane, thus increasinguptake by virtue of a depot effect (Illum et al. 2001; 2003; Davis etal. 1999; Bacon et al. 2000; van der Lubben et al. 2001; 2001; Lim etal. 2001). Chitosan has been tested as a nasal delivery system forseveral vaccines, including influenza, pertussis and diphtheria, in bothanimal models and humans (Illum et al. 2001; 2003; Bacon et al. 2000;Jabbal-Gill et al. 1998; Mills et al. 2003; McNeela et al. 2004). Inthese trials, chitosan was shown to enhance systemic immune responses tolevels equivalent to parenteral vaccination. In addition, significantantigen-specific IgA levels were also measured in mucosal secretions.Thus, chitosan can greatly enhance a nasal vaccine's effectiveness.Moreover, due to its physical characteristics, chitosan is particularlywell suited to intranasal vaccines formulated as powders (van der Lubbenet al. 2001; Mikszta et al. 2005; Huang et al. 2004).

Accordingly, in one embodiment, the present invention provides anantigenic or vaccine composition adapted for intranasal administration,wherein the composition includes antigen and optionally an effectiveamount of adjuvant. In preferred embodiments, the invention provides anantigenic or vaccine composition comprising Norovirus antigen such asNorovirus VLP, in combination with at least one delivery agent, such aschitosan, and at least one adjuvant, such as MPL®, CPGs, imiquimod,gardiquimod, or synthetic lipid A or lipid A mimetics or analogs.

The molecular weight of the chitosan may be between 10 kDa and 800 kDa,preferably between 100 kDa and 700 kDa and more preferably between 200kDa and 600 kDa. The concentration of chitosan in the composition willtypically be up to about 80% (w/w), for example, 5%, 10%, 30%, 50%, 70%or 80%. The chitosan is one which is preferably at least 75%deacetylated, for example 80-90%, more preferably 82-88% deacetylated,particular examples being 83%, 84%, 85%, 86% and 87% deacetylation.

Vaccine and Antigenic Formulations

The compositions of the invention can be formulated for administrationas vaccines or antigenic formulations. As used herein, the term“vaccine” refers to a formulation which contains Norovirus VLPs or otherNorovirus antigens of the present invention as described above, which isin a form that is capable of being administered to a vertebrate andwhich induces an immune response sufficient to induce a therapeuticimmunity to ameliorate an infection and/or to reduce at least onesymptom of an infection and/or to enhance the efficacy of another doseof VLPs or antigen. As used herein, the term “antigenic formulation” or“antigenic composition” refers to a preparation which, when administeredto a vertebrate, e.g. a mammal, will induce an immune response. As usedherein, the term “immune response” refers to both the humoral immuneresponse and the cell-mediated immune response. The humoral immuneresponse involves the stimulation of the production of antibodies by Blymphocytes that, for example, neutralize infectious agents, blockinfectious agents from entering cells, block replication of saidinfectious agents, and/or protect host cells from infection anddestruction. The cell-mediated immune response refers to an immuneresponse that is mediated by T-lymphocytes and/or other cells, such asmacrophages, against an infectious agent, exhibited by a vertebrate(e.g., a human), that prevents or ameliorates infection or reduces atleast one symptom thereof. Vaccine preparation is generally described inVaccine Design (“The subunit and adjuvant approach” (eds Powell M. F. &Newman M. J.) (1995) Plenum Press New York). The compositions of thepresent invention can be formulated, for example, for delivery to one ormore of the oral, gastro-intestinal, and respiratory (e.g. nasal)mucosa.

Where the composition is intended for delivery to the respiratory (e.g.nasal) mucosa, typically it is formulated as an aqueous solution foradministration as an aerosol or nasal drops, or alternatively, as a drypowder, e.g. for rapid deposition within the nasal passage. Compositionsfor administration as nasal drops may contain one or more excipients ofthe type usually included in such compositions, for examplepreservatives, viscosity adjusting agents, tonicity adjusting agents,buffering agents, and the like. Viscosity agents can be microcrystallinecellulose, chitosan, starches, polysaccharides, and the like.Compositions for administration as dry powder may also contain one ormore excipients usually included in such compositions, for example,mucoadhesive agents, bulking agents, and agents to deliver appropriatepowder flow and size characteristics. Bulking and powder flow and sizeagents may include mannitol, sucrose, trehalose, and xylitol.

In one embodiment, the Norovirus vaccine or antigenic formulation of thepresent invention may be formulated as a dry powder containing one ormore Norovirus genogroup antigen(s) as the immunogen, an adjuvant suchas MPL®, a biopolymer such as chitosan to promote adhesion to mucosalsurfaces, and bulking agents such as mannitol and sucrose. For example,the Norovirus vaccine may be formulated as 10 mg of a dry powdercontaining one or more Norovirus genogroup antigen(s) (e.g., Norwalkvirus, Houston virus, Snow Mountain virus), MPL® adjuvant, chitosanmucoadhesive, and mannitol and sucrose as bulking agents and to provideproper flow characteristics. The formulation may comprise about 7.0 mg(25 to 90% w/w range) chitosan, about 1.5 mg mannitol (0 to 50% w/wrange), about 1.5 mg sucrose (0 to 50% w/w range), about 25 μg MPL® (0.1to 5% w/w range), and about 100 μg Norovirus antigen (0.05 to 5% w/wrange).

Norovirus antigen may be present in a concentration of from about 0.01%(w/w) to about 80% (w/w). In one embodiment, Norovirus antigens can beformulated at dosages of about 5 μg, about 15 μg, and about 50 μg per 10mg dry powder formulation (0.025, 0.075 and 0.25% w/w) foradministration into both nostrils or about 10 μg, about 30 μg, and about100 μg (0.1, 0.3 and 1.0% w/w) for administration into one nostril. Theformulation may be given in one or both nostrils during eachadministration. There may be a booster administration 1 to 12 weeksafter the first administration to improve the immune response. Thecontent of the Norovirus antigens in the vaccine and antigenicformulations may be in the range of 1 μg to 100 mg, preferably in therange 1-500 μg, more preferably 5-200 μg, most typically in the range10-100 μg. Total Norovirus antigen administered at each dose will beeither about 10 μg, about 30 μg, or about 100 μg in a total of 20 mg drypowder when administered to both nostrils or 10 mg dry powder whenadministered to one nostril. Dry powder characteristics are such thatless than 10% of the particles are less than 10 μm in diameter. Meanparticle sizes range from 10 to 500 μm in diameter.

In another embodiment, the antigenic and vaccine compositions can beformulated as a liquid for subsequent administration to a subject. Aliquid formulation intended for intranasal administration would compriseNorovirus genogroup antigen(s), adjuvant, and a delivery agent such aschitosan. Liquid formulations for intramuscular (i.m.) or oraladministration would comprise Norovirus genogroup antigen(s), adjuvant,and a buffer, without a delivery agent (e.g., chitosan).

Preferably the antigenic and vaccine compositions hereinbefore describedare lyophilized and stored anhydrous until they are ready to be used, atwhich point they are reconstituted with diluent, if used in a liquidformulation. Alternatively, different components of the composition maybe stored separately in a kit or device (any or all components beinglyophilized). The components may remain in lyophilized form for dryformulation or be reconstituted for liquid formulations, and eithermixed prior to use or administered separately to the patient. For drypowder administration the vaccine or antigenic formulation may bepreloaded into an intranasal delivery device or topical (e.g., dermal)delivery patch and stored until used. Preferably, such delivery deviceand associated packaging would protect and ensure the stability of itscontents.

The lyophilization of antigenic formulations and vaccines is well knownin the art. Typically the liquid antigen is freeze dried in the presenceof agents to protect the antigen during the lyophilization process andto yield powders with desirable characteristics. Sugars such as sucrose,mannitol, trehalose, or lactose (present at an initial concentration of10-200 mg/mL) are commonly used for cryoprotection and lyoprotection ofprotein antigens and to yield lyophilized cake or powders with desirablecharacteristics. Lyophilized compositions are theoretically more stable.Other drying technologies, such as spray drying or spray freeze dryingmay also be used. While the goal of most formulation processes is tominimize protein aggregation and degradation, the inventors havedemonstrated that the presence of aggregated antigen enhances the immuneresponse to Norovirus VLPs (see Examples 3 and 4 in animal models).Therefore, the inventors have developed methods by which the percentageof aggregation of the antigen can be controlled during thelyophilization process to produce an optimal ratio of aggregated antigento intact antigen to induce a maximal immune response in animal models.

Thus, the invention also encompasses a method of making Norovirusantigen formulations comprising (a) preparing a pre-lyophilizationsolution comprising Norovirus antigen, sucrose, and chitosan, whereinthe ratios of sucrose to chitosan are from about 0:1 to about 10:1; (b)freezing the solution; and (c) lyophilizing the frozen solution for30-72 hours, wherein the final lyophilized product contains a percentageof said Norovirus antigen in aggregated form. The lyophilization mayoccur at ambient temperature, reduced temperature, or proceed in cyclesat various temperatures. For illustration purposes only, lyophilizationmay occur over a series of steps, for instance a cycle starting at −69°C., gradually adjusting to −24° C. over 3 hours, then retaining thistemperature for 18 hours, then gradually adjusting to −16° C. over 1hour, then retaining this temperature for 6 hours, then graduallyadjusting to +34° C. over 3 hours, and finally retaining thistemperature over 9 hours In one embodiment, the pre-lyophilizationsolution further comprises a bulking agent. In another embodiment, saidbulking agent is mannitol.

Appropriate ratios of sucrose and chitosan to yield desired percentagesof aggregation can be determined by the following guidelines. Apre-lyophilization mixture containing mass ratios of sucrose to chitosanin a range from about 2:1 to about 10:1 will yield a range of about 50%to 100% intact Norovirus antigen (i.e. 0% to 50% aggregated antigen)post-lyophilization depending on pre-lyophilization solutionconcentrations (see Example 13). Mass ratios of 0:1 sucrose to chitosanwill produce less than 30% of intact Norovirus antigen (i.e. greaterthan 70% aggregated antigen). Omission of both sucrose and chitosan anduse of only a bulking agent, such as mannitol, will produce less than10% intact antigen (i.e. greater than 90% aggregated antigen dependingon pre-lyophilization solution concentrations). Using these guidelines,the skilled artisan could adjust the sucrose to chitosan mass ratios andconcentrations in the pre-lyophilization mixture to obtain the desiredamount of aggregation necessary to produce an optimal immune response.

In addition, the inclusion of sucrose, chitosan, and mannitol in thepre-lyophilization solution has no negative effect on the stability ofthe intact Norovirus antigen over time, i.e. the ratio of aggregatedantigen/intact antigen in the formulation does not increase when storedas a dry powder for a period of about 12 months or greater (see Example10). Thus, this lyophilization procedure ensures stable formulationswith predictable and controllable ratios of aggregated to intactNorovirus antigen.

Methods of Stimulating an Immune Response

The amount of antigen in each antigenic or vaccine formulation dose isselected as an amount which induces a robust immune response withoutsignificant, adverse side effects. Such amount will vary depending uponwhich specific antigen(s) is employed, route of administration, andadjuvants used. In general, the dose administered to a patient, in thecontext of the present invention should be sufficient to effect abeneficial therapeutic response in the patient over time, or to inducethe production of antigen-specific antibodies. Thus, the composition isadministered to a patient in an amount sufficient to elicit an immuneresponse to the specific antigens and/or to alleviate, reduce, or curesymptoms and/or complications from the disease or infection. An amountadequate to accomplish this is defined as a “therapeutically effectivedose.”

For a substantially pure form of the Norovirus antigen, it is expectedthat each dose will comprise about 1 μg to 10 mg, preferably about 2-50μg for each Norovirus antigen in the formulation. In a typicalimmunization regime employing the antigenic preparations of the presentinvention, the formulations may be administered in several doses (e.g.1-4), each dose containing 1-100 μg of each antigen. The dose will bedetermined by the immunological activity the composition produced andthe condition of the patient, as well as the body weight or surfaceareas of the patient to be treated. The size of the dose also will bedetermined by the existence, nature, and extent of any adverse sideeffects that may accompany the administration of a particularcomposition in a particular patient.

The antigenic and vaccine formulations of the present invention may beadministered via a non-mucosal or mucosal route. These administrationsmay include in vivo administration via parenteral injection (e.g.intravenous, subcutaneous, and intramuscular) or other traditionaldirect routes, such as buccal/sublingual, rectal, oral, nasal, topical(such as transdermal and ophthalmic), vaginal, pulmonary, intraarterial,intraperitoneal, intraocular, or intranasal routes or directly into aspecific tissue. Alternatively, the vaccines of the invention may beadministered by any of a variety of routes such as oral, topical,subcutaneous, mucosal, intravenous, intramuscular, intranasal,sublingual, transcutaneous, subdermal, intradermal and via suppository.Administration may be accomplished simply by direct administration usinga patch, needle, catheter or related device, at a single time point orat multiple time points.

In a preferred embodiment, the antigenic and vaccine formulations of thepresent invention are administered to a mucosal surface. Immunizationvia the mucosal surfaces offers numerous potential advantages over otherroutes of immunization. The most obvious benefits are 1) mucosalimmunization does not require needles or highly-trained personnel foradministration, and 2) immune responses are raised at the site(s) ofpathogen entry, as well as systemically (Isaka et al. 1999; Kozlowski etal. 1997; Mestecky et al. 1997; Wu et al. 1997).

In a further aspect, the invention provides a method of eliciting an IgAmucosal immune response and an IgG systemic immune response byadministering (preferably intranasally or orally) to a mucosal surfaceof the patient an antigenic or vaccine composition comprising one ormore Norovirus antigens, at least one effective adjuvant and/or at leastone delivery agent.

The present invention also contemplates the provision of means fordispensing intranasal formulations of Norovirus antigens hereinbeforedefined, and at least one adjuvant or at least one delivery agent ashereinbefore defined. A dispensing device may, for example, take theform of an aerosol delivery system, and may be arranged to dispense onlya single dose, or a multiplicity of doses. Such a device would deliver ametered dose of the vaccine or antigenic formulation to the nasalpassage. Other examples of appropriate devices include, but are notlimited to, droppers, swabs, aerosolizers, insufflators (e.g. ValoisMonopowder Nasal Administration Device, Bespak UniDose DP), nebulizers,and inhalers. The devices may deliver the antigenic or vaccineformulation by passive means requiring the subject to inhale theformulation into the nasal cavity. Alternatively, the device mayactively deliver the formulation by pumping or spraying a dose into thenasal cavity. The antigenic formulation or vaccine may be delivered intoone or both nostrils by one or more such devices. Administration couldinclude two devices per subject (one device per nostril). Actual dose ofactive ingredient (Norovirus antigen) may be about 5-1000 μg. In apreferred embodiment, the antigenic or vaccine formulation isadministered to the nasal mucosa by rapid deposition within the nasalpassage from a device containing the formulation held close to orinserted into the nasal passageway.

The invention also provides a method of generating antibodies to one ormore Norovirus antigens, said method comprising administration of avaccine or antigenic formulation of the invention as described above toa subject. These antibodies can be isolated and purified by routinemethods in the art. The isolated antibodies specific for Norovirusantigens can be used in the development of diagnostic immunologicalassays. These assays could be employed to detect a Norovirus in clinicalsamples and identify the particular virus causing the infection (e.g.Norwalk, Houston, Snow Mountain, etc.). Alternatively, the isolatedantibodies can be administered to subjects susceptible to Norovirusinfection to confer passive or short-term immunity.

As mentioned above, the vaccine formulations of the invention may beadministered to a subject to treat symptoms of a Norovirus infection.Symptoms of Norovirus infection are well known in the art and includenausea, vomiting, diarrhea, and stomach cramping. Additionally, apatient with a Norovirus infection may have a low-grade fever, headache,chills, muscle aches, and fatigue. The invention encompasses a method ofinducing an immune response in a subject experiencing a Norovirusinfection by administering to the subject a vaccine formulation of theinvention such that at least one symptom associated with the Norovirusinfection is alleviated and/or reduced. A reduction in a symptom may bedetermined subjectively or objectively, e.g., self assessment by asubject, by a clinician's assessment or by conducting an appropriateassay or measurement (e.g. body temperature), including, e.g., a qualityof life assessment, a slowed progression of a Norovirus infection oradditional symptoms, a reduced severity of Norovirus symptoms orsuitable assays (e.g. antibody titer and/or T-cell activation assay).The objective assessment comprises both animal and human assessments.

EXAMPLES

The invention will now be illustrated in greater detail by reference tothe specific embodiments described in the following examples. Theexamples are intended to be purely illustrative of the invention and arenot intended to limit its scope in any way.

Example 1 Investigations into Immune Responses to Different NorovirusAntigen Forms

To investigate the efficacy of the vaccine formulations, mice wereimmunized intranasally (i.n.) with liquid suspension vaccine formulationby micropipette. Mice received only a single vaccine dose (prime).

For the experiment, three vaccine formulations were prepared. The first,referred to as 100% aggregate, was prepared by lyophilization of VLPsunder conditions that disrupt the native structure of the VLP and induceaggregation. The second, 100% intact, was prepared with rehydratedlyophilized placebo, spiked with 100% native monodisperse VLPs fromnon-lyophilized VLP stock. The third formulation, 50/50 Mix, is madeeither by mixing the previous two formulations at a ratio of 1:1, or bylyophilizing under conditions that yield ˜50% intact and 50% aggregatedVLPs. The structural state and concentration of the intact native VLPwas assayed by size exclusion high performance liquid chromatography(SE-HPLC) and ultraviolet (UV) absorbance. The total proteinconcentration (which includes the aggregate) of the formulations wasdetermined by quantitative staining of sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE)-resolved proteins. Percentaggregated/intact was calculated as the ratio of intact native VLP tototal protein.

TABLE 1 Mixtures shown below were prepared for Experiment 605.125, mousei.n. liquid vaccination. Group Chitosan Mannitol Sucrose MPL Norwalk VLPnumber (mg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL) 1 3.5 0.750 0.750 1.0 1.02 3.5 0.750 0.750 1.0 1.0 3 3.5 0.750 0.750 1.0 1.0 4 3.5 0.750 0.7501.0 0 Table 1. Prime for exp 605.125 (mouse i.n.) Values indicate finalconcentrations of the formulations. Dose: 20 μL per mouse, 10 μL pernare. Group 1, 100% Agg: rehydrated 100% aggregated VLP Group 2, 100%Intact: rehydrated lyophilized placebo, spiked with 100% intact VLPsfrom non-lyophilized VLP stock. Group 3, 50/50 mix: 1:1 mixture ofsolutions from Groups 1 and 2. Group 4, Naïve: rehydrated lyophilizedplacebo

This experiment measures the immune response in mice to differentNorovirus VLP formulations. Groups of mice (5 per group) were vaccinatedintranasally (i.n.) once with rehydrated dry powder formulations shownin Table 1. Animals vaccinated with VLP-containing formulations receivedthe same amount of total protein. 100% Agg (100% aggregated VLPprotein); 100% Intact (100% native, monodisperse VLPs); 50/50 Mix (1:1mixture of monodisperse and aggregated VLP); Naïve (no VLP protein). Onday 14 following i.n. immunization, mice were euthanized, the cervicallymph nodes and spleens were harvested, and a single cell suspension wasprepared for in vitro antigen-specific cell proliferation assays. Inthese assays the response of cervical lymph node cells or splenocyteswere assessed to determine immunogenic responses against the antigenfollowing in vivo immunization. Cervical lymph node cells or splenocyteswere restimulated with either native monodisperse VLPs (native VLP,black bars) or heat-denatured VLP protein (AVLP, white bars) and theextent of cellular proliferation from each antigen form (100% Agg, 100%Intact, 50/50 Mix, or naïve) was measured by tritiated thymidineincorporation as indicated on the ordinate axis (CPM) (FIG. 1, cervicallymph node cells; FIG. 2, splenocytes).

Example 2 In Vitro Antigen-Specific Proliferation Assay

To further investigate the potency of the vaccine formulations, micewere immunized intraperitoneally (i.p.) with liquid suspension vaccineformulation. Mice received only a single vaccine dose (prime).

Similar to Example 1, groups of mice (5 per group) were vaccinated, butthis time intraperitoneally (i.p.), once with rehydrated dry powderformulations shown in Table 2. Again, animals vaccinated withVLP-containing formulations received the same amount of total protein.100% Agg (100% aggregated VLP protein); 100% Intact (100% native, intactVLPs); 50/50 Mix (1:1 mixture of intact and aggregated VLP); Naïve (noVLP protein).

TABLE 2 Mixtures shown below were prepared for 605.127, mouse i.p.liquid immunization. Group Chitosan Mannitol Sucrose MPL Norwalk VLPnumber (mg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL) 1 7 1.475 1.475 0.0250.025 2 7 1.475 1.475 0.025 0.025 3 7 1.475 1.475 0.025 0.025 4 7 1.4751.475 0.025 0 Prime for exp 605.127 (mouse i.p.) Values indicate finalconcentrations of the formulations and are equivalent to a single 10 mgdelivery device. Dose: 1 mL per mouse i.p. Group 1, 100% Agg: rehydrated100% aggregated VLP Group 2, 100% Intact: rehydrated lyophilizedplacebo, spiked with 100% intact VLPs from non-lyophilized VLP stock.Group 3, 50/50 mix: rehydrated from lyophilized 50/50 intactVLP/Aggregate Group 4, Naïve: rehydrated lyophilized placeboIn this assay, response of different murine cells to VLPs following invivo immunization was measured. On day 14 following immunization, micewere euthanized, the spleens were harvested, and a single cellsuspension was prepared. Splenocytes were restimulated with eitherintact, native VLPs (native VLP, dotted bars) or heat-denatured VLPprotein (AVLP, white bars) and the extent of cellular proliferation fromeach antigen form (100% Agg, 100% Intact, 50/50 Mix, or naïve) wasmeasured by tritiated thymidine incorporation as indicated on theordinate axis (CPM) (FIG. 3). These data indicate that differentbiophysical forms of the VLPs prepared in the vaccine formulationselicit comparable T cell responses.

Example 3 VLP-Specific ELISPOT Assay

VLP-specific antibody-secreting cell (ASC) responses were measured frommice immunized intraperitoneally with different NV-VLP formulationsdescribed in Example 2. Groups of mice (5 per group) were vaccinatedi.p. once with rehydrated dry powder formulations shown in Table 2(Example 2). Animals vaccinated with VLP-containing formulationsreceived the same amount of total protein. 100% Agg (100% aggregated VLPprotein); 100% Intact (100% native, intact VLPs); 50/50 Mix (1:1 mixtureof intact and aggregated VLP); Naïve (no VLP protein). On day 14, themice were euthanized and the cervical lymph nodes were harvested. Thecervical lymph node cells were cultured overnight on native, intactVLP-coated ELISPOT plates and were developed for either IgG orIgA-specific ELISPOTS using the appropriate HRP-conjugated secondaryantibodies (FIG. 4). These data show that the three VLP antigenformulations all elicit an antigen-specific B cell response. The groupimmunized with 100% Agg VLPs exhibited the greatest immune response.

Example 4 VLP-Specific ELISA

Serum IgG levels were measured from mice immunized i.p. with differentNV-VLP formulations. Groups of mice (5 per group) were vaccinated i.p.once with rehydrated dry powder formulations shown in Table 2 (Example2). Animals vaccinated with VLP-containing formulations received thesame amount of total protein. 100% Agg (100% aggregated VLP protein);100% Intact (100% native, intact VLPs); 50/50 Mix (1:1 mixture of intactand aggregated VLP); Naïve (no VLP protein). On day 14, serum wascollected and assayed by ELISA for anti-VLP-specific serum IgG (FIG. 5).These data correlate with the results shown in Example 3, indicatingthat the three VLP antigen formulations all elicit an antigen-specific Bcell response. Again, the group immunized with 100% Agg VLPs showed thegreatest immune response.

Example 5 Vaccine Formulations in Rabbits

Formulations were administered intranasally (i.n.) in rabbits using theValois Monopowder Nasal Administration Device. The dry powderformulations are shown in Tables 3 and 4.

TABLE 3 Formulations described below were prepared for 605.129, rabbiti.n. dry powder (DP) vaccination. Prime formulations for exp 605.129(Rabbit i.n.) (final amounts for DP vaccines). Norwalk Chitosan MannitolSucrose MPL VLP Group (mg/ (mg/ (mg/ (mg/ (mg/ number 10 mg DP) 10 mg)10 mg) 10 mg) 10 mg) 1 7 1.475 1.475 0.025 0.025 2 7 1.475 1.475 0.0250.025 3 7 1.475 1.475 0.025 0.025 4 7 1.475 1.475 0.025 0 Valuesindicate final concentrations of the formulations based on a singledevice (10 mg DP) which is ½ total dose. Dose: 20 mg DP per animal, 10mg per nare. Group 1, 100% Agg: 100% aggregated lyophilized VLP Group 2,100% Intact: 100% intact lyophilized VLP Group 3, 50/50 mix: 50/50intact/aggregate lyophilized VLP (not a mixture of 1 & 2) Group 4,Naïve: placebo

TABLE 4 Formulations shown below were prepared for 605.129, rabbit i.n.dry powder (DP) vaccination. Boost formulations for exp 605.129 (Rabbiti.n.) (final amounts for DP vaccines). Norwalk Chitosan Mannitol SucroseMPL VLP Group (mg/ (mg/ (mg/ (mg/ (mg/ number 10 mg DP) 10 mg) 10 mg) 10mg) 10 mg) 1 7 1.475 1.475 0.025 0.025 2 7 0 2.95 0.025 0.025 3 7 1.4751.475 0.025 0.025 4 7 1.475 1.475 0.025 0 Values indicate finalconcentrations of the formulations based on a single device (10 mg DP)which is ½ total dose. Dose: 20 mg DP per animal, 10 mg per nare. Group1, 100% Agg: 100% lyophilized aggregated VLP Group 2, 100% Intact: 100%intact** VLP* Group 3, 50/50 mix: 50/50 intact/aggregate lyophilized VLP(not a mixture of 1 & 2) Group 4, Naïve: lyophilized placebo *Formulatedwithout mannitol to increase amount of intact VLP post lyophilization.**Preparation yielded only ~80% intact VLP.

Example 6 Potency Assay of Norovirus Vaccine Formulation in Mice

Female C57B16 mice were immunized intraperitoneally (i.p.) on day 0 withdifferent dilutions of a reconstituted Norwalk VLP dry powder vaccine(containing Norwalk VLP, MPL and chitosan). Each animal was injectedwith 100 μL of the formulations indicated. Serum was collected weeklyand serum anti-VLP IgG measured by ELISA. Values for serum collected 3weeks following immunization are shown in FIG. 6.

The value for each individual mouse is represented, with bars indicatingthe group mean. Serum anti-VLP IgG values correlated with the dose ofvaccine indicated. This experimental design has been refined anddeveloped as a potency assay required for the release of GMPmanufactured vaccines for human clinical trials (FIG. 6).

Example 7 Potency of Liquid Vs. Reconstituted Norovirus Formulations inMice

Female C57B16 mice were immunized i.p. on day 0 with formulations thatcontained chitosan, mannitol, MPL, and various concentrations of NorwalkVLP (Table 5) in a volume of 100 μL. An internal standard curve wasgenerated (groups 1-5) by solubilizing 10 mg/mL of dry powder matrix(mannitol, MPL, and chitosan) in purified water and adding the specifiedamounts of liquid Norwalk VLP. In contrast, the GMP VLP lots werepreviously lyophilized and then solubilized in 1.0 ml of purified water(groups 6-8). Serum was collected from mice on days 14, 21 and 30, andserum anti-Norwalk VLP IgG was measured by ELISA.

TABLE 5 Liquid and Reconstituted Norwalk Formulations used to immunizemice (i.p.). Calculated Potency Group Treatment 95% CI Potency Min Max 15 μg VLP in Placebo 0.173 58.0 39.0 86.3 2 2.5 μg VLP in Placebo 0.19223.3 15.0 36.3 3 1.25 μg VLP in Placebo 0.182 11.2 7.4 17.0 4 0.63 μgVLP in Placebo 0.287 5.4 2.8 10.4 5 0.31 μg VLP in Placebo 0.114 3.8 2.94.9 6 2.5 μg GMP lot 0.276 11.3 6.0 21.3 7 7.5 μg GMP lot 0.221 96.858.2 161.0 8 25 μg GMP lot 0.147 113.6 80.9 159.5

The relative potency for each formulation was calculated using thefollowing formula: Inv Log (Ave.−Y intercept/slope). Potency is plottedagainst VLP concentration in the formulations and reported in relationto the standard curve generated using known amounts of VLP spiked intothe matrix background (FIG. 7). The results shown are representative of3 separate serum collection time points. These data indicate that theNorwalk VLP formulation reconstituted from dry powder has an overallhigher potency than the liquid formulations.

Example 8 Potency of Dry Powder Formulation in Rabbits

Forty-three female New Zealand White rabbits were intranasally (i.n.)immunized using the Valois Monopowder Nasal Administration Device witheither 5 μg (Low) or 25 μg (Hi) of Norwalk VLPs±MPL and ±chitosanformulated into dry powders. One group received the Hi dose of VLPs andMPL formulated as a liquid and administered intramuscularly (i.m.).Rabbits were vaccinated on days 0 and 21. MPL, when used, was used atthe same dose as the VLPs (i.e., 5 μg Norwalk VLPs and 5 μg MPL).Chitosan, when used, was 7 mg/dose.

Serum IgG specific for the Norwalk VLPs (as determined by ELISA) isshown in FIG. 8. Mean values for each treatment groups are shown for day21 (left panel, collected just prior to administration of the boosterimmunization) and day 42 (right panel). Values are reported in U/mL ofVLP-specific IgG, with 1 U approximating 1 μg. Standard deviations areindicated by bars. All treatment groups had 6 animals, except thenegative control group (3 rabbits) and the intramuscularly immunizedgroup (4 animals). These data show that generally the higher VLP doseresults in greater serum anti-VLP IgG levels. Chitosan, in particular,enhances responses to intranasal vaccines. The i.m. immunized groupshowed the greatest responses. However, VLP-specific IgG levels in theintranasally immunized groups were also quite robust.

Example 9 Potency of Liquid Vs. Dry Norovirus Formulations GivenIntranasally in Rabbits

Female New Zealand White rabbits were intranasally immunized using theValois Monopowder Nasal Administration Device with 50 μg of NorwalkVLPs+50 μg MPL+14 mg chitosan formulated into either a dry powder or aliquid. The vaccine content was identical, except for the physicalstate. Immunizations were on days 0 and 21 (weeks 0 and 3), with serumcollected prior to the boost at 3 weeks, and again at 6 weeks followingthe initial vaccination. Serum IgG specific for Norwalk VLPs wasmeasured by ELISA, and the results are shown in FIG. 9.

Group means are indicated, with the bars representing standarddeviations. The dry powder immunization group had 6 rabbits, and theliquid immunization group had 10 rabbits. Eight negative control rabbitsare represented. Little difference was seen between the liquid and drypowder immunization groups at 3 weeks; however, at 6 weeks following theinitial immunization, rabbits immunized with the dry powder formulationhad superior serum anti-VLP IgG responses compared to the liquidimmunization group.

Example 10 Stability of Norovirus Dry Powder Formulations

To investigate the stability of the dry powder VLP formulation, bulkdrug product was prepared by mixing (per 10 mg drug product) 25 μg of aGenogroup I VLP in solution with 25 μg MPL, 700 μg chitosan glutamate,1.475 mg mannitol, and 1.475 mg sucrose. The solution was lyophilized,blended with an additional 6.3 mg chitosan glutamate (per 10 mg drugproduct), filled into Bespak unidose devices at a nominal 10 mg of drypowder, and stored in sealed foil pouches with desiccant capsules. TotalVLP content was measured using Imperial stained SDS-PAGE and scanningdensitometry, while size exclusion chromatography (SEC) was used toquantify intact VLP content. These measurements indicated that, withinexperimental error, no change in either total or intact VLP wasdetectable over the 12 month period (FIG. 10). Assuming that the lowerVLP protein recovery by SEC, when compared to SDS-PAGE results, was dueto aggregation, the calculated % aggregate did not increase with timebut rather remained constant or decreased throughout the 12 months ofstorage. One of the more common stability issues with proteins isincreased aggregation with storage. Based on the results in FIG. 10, itcan be concluded that the formulation results in a stable percentage ofintact VLPs allowing the product to be manufactured and used over atleast a one year period.

Example 11 Multiple Norovirus Antigens

Eight C57B1/6 mice (female, 9 weeks of age) were immunizedintraperitoneally (IP) on days 0 and 14 with 2.5 μg Norwalk VLPformulated with 0.7 mg chitosan, 2.5 μg MPL and 0.3 mg mannitol broughtto 0.1 mL with water. Two control mice were immunized with saline. Ondays 28 and 49, they were immunized again IP with 2.5 μg Norwalk VLP+2.5μg Houston VLP formulated with 0.7 mg chitosan, 2.5 μg MPL and 0.3 mgmannitol brought to 0.1 mL with water. The control mice again receivedsaline. Serum samples were collected weekly beginning on week 5 (day 35)and analyzed by ELISA for reactivity with Norwalk VLPs or Houston VLPs.The time of boost with the Norwalk only mixtures are indicated by thethin arrows, and the Norwalk+Houston VLP mixtures are indicated by thickarrows. Individual serum IgG responses specific for Norwalk VLPs (toppanel) or Houston VLPs (bottom panel) in U/mL (with 1 U approximating 1μg of IgG) are shown. Means are indicated by bars. Note that the Y-axisscales are different, as the anti-Norwalk responses were much morerobust due to two previous immunizations on days 0 and 14 (weeks 0 and2). However, the responses against Houston VLPs are quite robust, with alarge increase appearing in the second week after the boost. These datademonstrate that specific immune responses can be generated againstdifferent antigenic strains of Norovirus VLPs in the same immunizingmixture. (FIG. 11).

Example 12 Immune Response to Different Norovirus Antigens

Female C57B16 mice were immunized intraperitoneally (IP) on days 0 and14 with 25 μg Norwalk VLP, 25 μg Houston VLP, or a combination of 25 μgof each Norwalk and Houston VLP. Serum was collected weekly and serumanti-VLP IgG measured by ELISA. Values for serum collected 4 weeksfollowing immunization are shown in FIG. 12.

VLP content of the immunizations is indicated on the X axis. The valuefor each individual mouse is represented, with bars indicating the groupmean. Antibody levels are represented in U/mL, with 1 U approximating 1μg of serum IgG. Values in the left panel were determined using NorwalkVLPs as the capture agent, while Houston VLPs were used to coat ELISAplates in order to measure the values on the right panel. These datashow that immunization with Norwalk VLP does not lead to serumantibodies that are able to recognize Houston VLPs, or vice versa.

Example 13 Mixtures of Sucrose and Chitosan Preserve Norovirus VLPStructure in Dry Powder Formulations

The following experiments examined the effects of sucrose, chitosan, andmannitol, alone or in combination, in pre-lyophilization solutions onthe native Norwalk VLP quaternary structure during lyophilization. Table6 is a composite of several experiments showing pre-lyophilizationsolution concentrations of the constituents of interest, the totalvolume of the mixture, and the corresponding mass ratios. All solutionswere manually swirled and gently vortexed to homogeneity, then shellfrozen in liquid nitrogen and lyophilized external to the unit usingside-arm vessels for times ranging from about 30 to 60 hours.

TABLE 6 Pre-lyophilization solution mixtures used for testing theeffects of different concentrations and combinations of sucrose,chitosan glutamate (chitosan) and mannitol on the structure of thequaternary structure the Norwalk VLP. Mass equivalents S = sucroseSolution concentrations of constituents pre- Total C = chitosanExperiment lyophilization (mg/mL) Volume M = mannitol and Sample SucroseChitosan Mannitol VLP (protein) (mL) S C M LE1 0 0 100 0.83 0.30 0 0 1LE2 0 0 75.0 0.62 0.40 LE3 0 0 50.0 0.42 0.60 LE4 0 0 25.0 0.21 1.20 LE50 0 10.0 0.08 3.00 LG1-LG3 0 7.83 0 0.20 1.28 0 1 0 LG4-LG6 0 5.06 00.13 1.98 LG7-LG9 0 2.09 0 0.05 4.78 LG10 19.32 1.93 0 0.05 5.18 10 1 0LG11 10.05 2.01 0 0.05 4.98 5 1 LG12 5.13 2.05 0 0.05 4.88 2.5 1 LG139.52 0.00 0 0.09 2.63 1 0 LJ1-LJ2 5.29 2.51 0 0.09 2.79 2 1 0 LJ3-LJ44.17 1.98 0 0.07 3.54 LJ5-LJ6 3.65 1.73 0 0.06 4.04 LJ7-LJ8 2.93 1.39 00.05 5.04 LJ9-LJ10 5.25 2.49 0 0.09 2.81 LJ11-LJ12 4.14 1.97 0 0.07 3.56LJ13-LJ14 3.63 1.72 0 0.06 4.06 LIG1d-Sa 2.98 1.42 0.00 1.12 4.94 2 1 0LIG1d-S1 12.89 6.12 0.00 1.12 2.29 2 1 0 LIG1d-S2 12.26 5.82 12.26 0.672.41 1 0.5 1 LIG1d-Sb 2.95 1.40 2.95 0.67 5.00 1 0.5 1 LIG1d-S3 29.320.00 29.32 0.83 1.01 0 0 1

Table 7 shows the results from size exclusion-high performance liquidchromatography (SE-HPLC) analysis of the lyophilized samples shown inTable 6. Lyophilized samples were reconstituted with water and analyzedby SE-HPLC. Unprocessed NV-VLPs, analyzed concurrently, were used as areference standard to quantify the NV-VLP content of the reconstitutedtest samples. Both UV and fluorescence detectors were used forquantification (data shown are from the fluorescence detector). TheSE-HPLC was conducted using a Superose™ 6 10-300 column, with mobilephase consisting of 10 mM sodium phosphate, 10 mM citric acid, pH 5, and500 mM NaCl, at a flow rate of 0.5 mL/min. Protein concentrations werequantified using integrated areas of elution peaks. “VLP” is the peakthat eluted at about 15 min from the column, and any preceding shoulderand/or peak tail within the approximate elution time window of thereference standard NV-VLP analyzed concurrently. The VLP fragment thatelutes from the column at around 32 min is a highly stable singlespecies that results from destabilization and consequent disassembly ofthe VLP. Intermediate and smaller fragments were not observed.

The results show that combinations of sucrose and chitosan produced awide range of native monodisperse NV-VLP recoveries including thehighest (approximately 100% recovery) post-lyophilization (samplesLG10-LG12). Moreover, the NV-VLP elution peak shapes from these sampleswere identical to the unprocessed NV-VLP reference standard indicatinghigh preservation of native structure. Samples containing sucrose onlyexhibited peak shapes similar to the reference standard, though NV-VLPrecoveries were lower (approx. 60% recovery (sample LG13) Samples thatcontained only mannitol resulted in nearly completely aggregated VLPs(samples LE1-LE6 and LIG1d-S3). The deleterious effects of mannitol onNV-VLP structure were counteracted by the presence of chitosan andsucrose (samples LIG1d-S2 and LIG1d-SB).

TABLE 7 Experiment and sample identification, and results for testingthe effect of sucrose, chitosan, and mannitol or combinations thereof onstability of NV-VLP structure during freezing and lyophilization.Measured SE-HPLC mean protein Mean percent concentration values of andpeak recovered protein Mass elution time as percent of equivalents VLPtheoretical S = sucrose Theoretical “VLP” Fragment Total C = chitosanExperiment VLP conC ~15 min ~32 min protein “VLP” M = mannitol andSample (mg/mL) N (mg/mL) (mg/mL) (%) (%) S C M LE1-LE5 0.25 5 0.02 0.1256.0 6.3 0 0 1 LG1-LG9 0.25 9 0.06 0.00 24.0 24.0 0 1 0 LG10 0.25 1 0.250.00 101 101 10 1 0 LG11 0.25 1 0.25 0.00 101 101 5 1 0 LG12 0.25 1 0.250.00 100 100 2.5 1 0 LG13 0.25 1 0.16 0.00 65 65 1 0 0 LJ1-LJ14 0.25 140.22 0 85.4 85.4 2 1 0 LIG1d-S1 0.25 1 0.21 0 88 88 2 1 0 LIG1d-Sa 0.251 0.12 0 50 50 2 1 0 LIG1d-S2 0.25 1 92 0 92 92 1 0.5 1 LIG1d-Sb 0.25 160 0 60 60 1 0.5 1 LIG1d-S3 0.25 1 <1 <1 <1 <1 0 0 1

Example 14 Induction of Norovirus-Specific Long-Lived Plasma Cells andMemory B Cells in Mice Immunized Intranasally A. Norwalk VLP-SpecificLong-Lived Plasma Cells

BALB/c mice were immunized intranasally with Norovirus VLPs and anadjuvant. Naïve controls were administered the adjuvant alone. At 114days after immunization, spleen, cervical lymph nodes, and bone marrowwere harvested from both groups of mice. On the day of harvesting thetissues (day 0), cells were assayed using an ELISPOT assay for thepresence of antigen-specific antibody-secreting cells (ASCs). Theresults are presented in FIG. 13A-C for the different tissues. Thedetection of immunoglobulins (IgG, IgA, and IgM) in these tissuesindicates the presence of Norovirus-specific long-lived plasma cells.

B. Norwalk VLP-Specific Memory B Cells

An in vitro assay was developed to detect the presence of NorwalkVLP-specific memory B-cells from mice immunized intranasally withNorwalk VLPs. Various lymphoid tissues or whole blood (peripheral bloodmononuclear cells, splenocytes, lymph node cells, etc.) can serve as thesource of cells that can be assayed for the presence of memory B-cellsusing this assay.

In this experiment, the spleen was harvested and processed fromimmunized and naïve animals (controls), and splenocytes were culturedfor four days in the presence or absence (controls) of Norwalk VLPs (20μg/ml). An initial VLP-specific ELISPOT assay was performed on the dayof tissue harvest (day 0) to establish background levels of ASCs (seeSection A above). After four days in culture the cells were harvestedand assayed again in an ELISPOT assay to quantify the number ofVLP-specific ASCs. The difference in VLP-specific ASC numbers betweenthe day 0 and the day 4 assays represent the antigen-specific memoryB-cell population. The results of this experiment are shown in FIGS. 14Aand B.

Example 15 Norovirus Memory B Cell Responses in Rabbits

Two female New Zealand White rabbits were immunized intranasally with adry powder formulation consisting of 25 μg Norwalk VLP, 25 μg MPL, 1.5mg mannitol, 1.5 mg sucrose, and 7.0 mg chitosan per 10 mg of dry powderloaded into Valois Mark 4 intranasal delivery devices. The two rabbitsreceived a total of three immunizations at 14 day intervals. For theseexperiments, a non-immunized female rabbit was used as a naïve control.

A. Collection and Processing of Rabbit Tissues

Peripheral blood mononuclear cells (PBMCs): Whole blood (˜50 mL) wasobtained from rabbits in collection tubes containing EDTA to preventcoagulation. The whole blood was diluted 1:3 with sterile D-PBS and ˜35mL of diluted whole blood was layered onto 15 mL of LympholyteSeparation Medium in a sterile 50-mL centrifuge tube. The tubes werecentrifuged at 800×g for 20 minutes at room temperature. The buffy coatlayer containing the PBMCs was carefully removed using a sterile 5 mLpipette and the cells were washed twice with D-PBS. If necessary,contaminating red blood cells were removed by ACK lysis. The cells wereresuspended in RPMI-1640-10% FBS (1640-C) and counted in a hemocytometerusing a Trypan exclusion method.

Mesenteric lymph node cells: The lymph nodes were aseptically collectedfrom each rabbit separately following euthanasia. The tissues weremaintained in a sterile plastic Petri dish containing ˜10 mL ofRPMI-1640-No Serum (1640-NS). The lymph nodes were pressed through asterile mesh screen using a sterile pestle to disperse the tissue andobtain a single cell suspension of lymph node cells. The cells werecollected, washed twice with 1640-NS, and finally filtered through asterile 70 μm filter to remove clumps and debris. The cells wereresuspended in 1640-C and counted in a hemocytometer using a Trypan blueexclusion method.

Splenocytes: Spleens were aseptically obtained from each rabbitfollowing euthanasia. The spleens were placed in sterile Petri dishescontaining approximately 10 mL of 1640-NS. Using a sterile 22-guageneedle and syringe the media was repeatedly injected into the tissue todisrupt the splenic capsule and elaborate the cells. Sterile forcepswere then used to tease apart the remaining tissue fragments. Thecontents of the Petri dish were transferred to a sterile centrifuge tubeand the cell suspension and disrupted splenic tissue was allowed to sitfor 6-8 minutes to allow for the settling of large tissue fragments. Thesingle cell suspension was transferred to a second sterile centrifugetube and the cells were washed once with 1640-NS. The red blood cells inthe splenocyte prep were removed by an ACK lysis (8 mL ACK buffer, 8minutes, room temperature) and the cells were washed one more time with1640-NS and finally filtered through a sterile 70 μm filter to removeclumps and debris. The final cell pellet was resuspended in1640-Complete and counted in a hemocytometer using a Trypan blueexclusion method.

Bone marrow cells: The tibia bones in the lower legs were removed fromindividual rabbits following euthanasia. To remove the bone marrow cellsthe ends of the bones were aseptically cut off using a bone saw and thecontents of the bone were flushed out by repeated injections of 1640-NSmedium. The bone marrow cells were pipetted up and down repeatedly tobreak up and disperse clumps of cells. The cells were washed once with1640-NS; the red blood cells were lysed with ACK, and the cells werewashed one more time with 1640-NS. Finally, the cells were filteredthrough a sterile 70 μm filter to remove clumps and debris. The finalcell pellet was resuspended in 1640-Complete and counted in ahemocytometer using a Trypan blue exclusion method.

B. ELISPOT Assays

Following pre-wetting and washing, 96-well Millipore PVDF filter plateswere coated with a sterile solution of native Norwalk VLPs at aconcentration of 40 μg/ml in a final volume of 50 μl/well. The plateswere incubated overnight at 4° C., washed with D-PBS, and blocked withthe addition of 1640-C. Mesenteric lymph node cells, splenocytes, andbone marrow cells from the immunized rabbits and from the naïve controlrabbit were added to the wells at varying concentrations (1×10⁶, 5×10⁵,2×10⁵, and 1×10⁵ cells/well) and the plates were incubated overnight at37° C. The plates were washed thoroughly with PBS-Tween and secondaryreagents specific for rabbit IgG and IgA were added to the wells andincubated for an additional 2 hours at room temperature. Followingextensive washing the plates were developed with DAB chromagen/substrateand read in an ELISPOT plate reader. Spots appearing on wells from naïvecontrol animals were subtracted from the experimental groups. The datais expressed as Norwalk VLP-specific antibody-secreting cells (ASCs) andis normalized per 1×10⁶ cells.

C. Norwalk VLP-Specific Memory B-Cell Assay

Isolated lymphoid cells from the various tissues described above wereresuspended in 1640-C medium in the presence of Norwalk VLPs (10 μg/mL)at a density of 5×10⁶ cells per mL. The cells were incubated in 24-wellplates in 1-mL volumes for four days at 37° C. VLP-specific ELISPOTassays were performed on these cells at the time of culturing. Afterfour days in culture the cells were harvested, washed twice with 1640-NSmedium, resuspended in 1640-Complete, and counted in a hemocytometerusing a Trypan blue exclusion method. The cells were tested once againin a Norwalk VLP-specific ELISPOT assay. The data obtained from theELISPOT assays performed on the day of tissue harvest is referred to asday 0 (background) ASC activity. Any spots detected at the day 0 timepoint are assumed to be actively-secreting plasma cells or long-livedplasma cells (LLPCs). The data obtained from the ELISPOT assay performedon the 4-day cultured cells is referred to as day 4 ASC activity, andthe memory B-cell activity is represented by the difference between day4 ASC activity and day 0 ASC activity.

D. Norwalk VLP-Specific Memory B-Cells are Present in the PeripheralBlood of Intranasally Immunized Rabbits

Whole blood was obtained from two immunized rabbits (RB735, RB1411) 141days following the last of three intranasal immunizations with a drypowder formulation vaccine containing Norwalk VLPs as described above.Blood was also obtained from an non-immunized, naïve rabbit. The bloodwas processed to obtain peripheral blood mononuclear cells (PBMCs) andthe PBMCs were placed in a Norwalk VLP-Specific memory B-Cell assay(section C above). The results are shown in FIG. 15. The left panelshows results of the initial ELISPOT assay at the time of tissue harvest(day 0 ASCs). The right panel shows the results of the ELISPOT assayafter 4 days in culture with Norwalk VLPs (day 4 ASCs).

The day 0 ELISPOT results (FIG. 15, left panel) illustrate that thereare no VLP-specific plasma cells remaining in the peripheral bloodapproximately 140 days after the last boost with Norwalk VLP dry powdervaccine. The right panel of FIG. 15 shows the ELISPOT assay results fromPBMCs cultured for four days in vitro with Norwalk VLPs. In the twoimmunized rabbits, a significant number of PBMCs, presumably asubpopulation of memory B-cells, have matured into active IgG-secretingNorwalk VLP-specific plasma cells. Although assays for IgA-secretingmemory B-cells were conducted, only IgG-secreting memory B-cells weredetected in the PBMC population. As expected, the naïve animal showed noantigen-specific memory B-cells. Thus, VLP-specific memory B-cells werefound in the peripheral circulation of rabbits 140+ days following thelast of three intranasal immunizations.

E. Norwalk VLP-Specific Memory B-Cells are Present in the Spleen ofIntranasally Immunized Rabbits

Splenocytes were obtained from the spleens of the two vaccine immunizedrabbits and the non-immunized control rabbit. Norwalk VLP-specificmemory B-cell assays (described above) were performed on these cells andthe results are shown in FIG. 16. As observed for the PBMC populationthe day 0 ELISPOT assay shows that there are no antigen-specific plasmacells present in the spleen (FIG. 16, left panel). However, following afour day in vitro incubation with Norwalk VLPs, IgG-secreting NorwalkVLP-specific memory B-cells are apparent in the splenocyte population.Thus, the spleen represents one site for the migration of memory B-cellsfollowing intranasal immunization.

F. A Population of Norwalk VLP-Specific Long-Lived Plasma Cells is Foundin the Bone Marrow but No Memory B-Cells are Present

Bone marrow cells were obtained from the tibias of the experimentalrabbits and assayed for the presence of long-lived plasma cells andmemory B-cells. The results are presented in FIG. 17. The left panel ofFIG. 17 shows that rabbit 1411 still had a significant population ofantigen-specific plasma cells in the bone marrow. Plasma cells thatmigrate to the bone marrow and reside there for a significant period oftime following immunization are referred to as long-lived plasma cells(LLPCs). Rabbit 735 did not show a high number of LLPCs. No LLPCs werefound in the bone marrow of the naïve rabbit. The bone marrow cells werecultured in a memory B-cell assay and re-tested for the presence ofmemory B-cells. The right panel of FIG. 17 shows that there areessentially no antigen-specific memory B-cells present in the bonemarrow. Thus, long-lived plasma cells migrate to the bone marrow but nomemory B-cells are found there.

G. Both IgG-Secreting and IgA-Secreting Norwalk VLP-Specific MemoryB-Cells are Present in the Mesenteric Lymph Nodes of IntranasallyImmunized Rabbits

The mesenteric lymph nodes were obtained from all of the experimentalrabbits and the isolated cells were assayed for LLPCs and memoryB-cells. The results from this assay are shown in FIG. 18A. As with mostof the lymphoid tissue analyzed, except bone marrow, no LLPCs (FIG. 18Aleft panels) were found in the mesenteric nodes. Following in vitroincubation with Norwalk VLPs, a very high number of IgG-secretingVLP-specific memory B-cells were evident in the mesenteric lymph nodepopulation (FIG. 18A, right panel). The numbers of memory B-cellsobserved in the mesenteric lymph nodes were significantly higher thanthose observed for the other lymphoid tissues assayed.

Numerous researchers have shown that immunization at a mucosal inductivesite, such as the nasal passages or the gut, is capable of eliciting aso-called mucosal immune response. This response has generally beencharacterized by the presence of IgA+ B-cells and IgA-secreting plasmacells localized in the mucosal lymphoid tissue. For this reason themesenteric lymph node cells were also assayed for the presence ofIgA-secreting LLPCs or memory B-cells. The results from these assays areshown in FIG. 18B. Once again, no IgA+ LLPCs were found in themesenteric lymph node population (FIG. 18B, left panel). However,IgA-secreting memory B-cells were detected in this tissue (FIG. 18B,right panel). Thus, intranasal immunization with a dry powder NorwalkVLP vaccine formulation elicited a mucosal immune response that resultedin the migration of both IgG+ and IgA+ antigen-specific memory B-cellsto the gut-associated lymphoid tissue. The production ofantigen-specific memory B cells induced by immunization with the Norwalkvaccine formulation is a possible indicator of vaccine effectiveness.The presence of memory B cells is one marker of long-lasting immunity.

H. VLP-Specific CD4+ Memory T Cells

Splenocytes harvested from immunized rabbits were restimulated withintact Norwalk VLPs and the extent of cellular proliferation wasmeasured by tritiated thymidine incorporation as indicated on theordinate axis (CPM) (FIG. 19). The left panel shows cellularproliferation of an unfractionated population of splenocytes, while theright panel shows cellular proliferation of CD4+ T cells.

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components are within the scope of the invention. Indeed, variousmodifications of the invention, in addition to those shown and describedherein, will become apparent to those skilled in the art from theforegoing description and accompanying drawings using no more thanroutine experimentation. Such modifications and equivalents are intendedto fall within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

Citation or discussion of a reference herein shall not be construed asan admission that such is prior art to the present invention.

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What is claimed is:
 1. A method of generating an immune response to atleast two Norovirus strains by administering to a patient an antigenicformulation comprising a combination of two or more monovalent NorovirusVLPs of different Norovirus genotypes, or an antigenic formulationcomprising multivalent VLPs containing capsid proteins from two or moredifferent Norovirus genotypes, such that the combination of VLPs withinthe formulation does not block the immunogenicity of each VLP type. 2.The method of claim 1, wherein immunity is generated against at leastone Norovirus strain having a genotype that is different than thegenotypes of said VLPs in said formulation.
 3. The method of claim 1,wherein said two or more Norovirus VLPs are of different genogroups. 4.The method of claim 3, wherein said genogroups are selected from thegroup consisting of GI, GII, GIII and GIV.
 5. The method of claim 1,wherein said monovalent Norovirus VLPs are derived from genogroup I orgenogroup II viral sequences.
 6. The method of claim 5, wherein saidmonovalent Norovirus VLPs are derived from genotypes I.1 and II.4. 7.The method of claim 1, wherein the antigenic formulation is administeredmucosally or parenterally.
 8. The method of claim 7, wherein mucosaladministration is accomplished intranasally.
 9. The method of claim 7,wherein parenteral administration is accomplished intramuscularly. 10.The method of claim 1, wherein the antigenic formulation includes one ormore adjuvants selected from the group consisting of toll-like receptoragonists and aluminum salts.
 11. The method of claim 1, wherein theantigenic formulation comprises both a toll-like receptor agonist and analuminum salt.
 12. The method of claim 1, wherein said toll-likereceptor agonist is monophosphoryl lipid A (MPL).